Functional substrates for growth of culinary and medicinal mushrooms

ABSTRACT

Methods of increasing the amount of one or more functional compounds in medicinal mushroom biomass and mushroom mycelia by growing the mushrooms on a substrate that is high in the functional compound or compounds of interest is provided. More specifically, a method of increasing the amount of anthocyanins in mushrooms is provided. Mushrooms are grown on a substrate rich in anthocyanins, such as purple corn or other suitable red, purple or black-pigmented substrate, and incorporate the anthocyanins during growth. Mushrooms grown by this method, as well as methods of improving the growth rate and potency of mushrooms, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/029,634, filed Jan. 5, 2005, which claims the benefit ofU.S. Provisional Application No. 60/534,334, filed Jan. 5, 2004, whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to substrates for use in growing mushrooms andmethods of increasing the growth rate of mushrooms. Specifically,functional substrates having desirable compounds can be used to growmushrooms, to increase the levels of these compounds in mushrooms and toimprove the growth rate of the mushrooms. Additionally, functionalsubstrates increase the potency of naturally occurring compounds alreadypresent in the mushrooms.

2. Background Information

In the commercial method of producing mushrooms, a suitably preparedsubstrate such as corn, rice, millet or rye is impregnated with mushroomspores or previously collected mushroom mycelia. Under sterile labconditions, the spores or mycelia are injected into the substrate, whichhas been prepared by soaking the grain in water and sterilizing it.Mycelia are the filamentous hyphae of a mushroom that collect water andnutrients to enable mushrooms to grow. The grains are incubated topromote full colonization of the mycelia, at which point themycelia-laced grains are known as “spawn”. Spawning is usually done in aplurality of individual spawn bags. The substrate provides the nutrientsnecessary for mycelium growth. The mycelium-impregnated substrate isthen allowed to develop under carefully controlled conditions oftemperature and moisture, until the hyphae of the mycelium havepermeated the substrate. This process usually takes anywhere from threeto four weeks for the mycelium to fully colonize the spawn bag. Thespawn bag is allowed to continue to grow until the mycelium enrichedproduct can be harvested between four to eight weeks from the beginningof the process. Typically, mushroom growers purchase spawn or grow itthemselves from agar plates, as will be known to one skilled in the art.In the commercial production of medicinal mushrooms, the spawn bag isthe final product, which is then sold or the contents processed into drypowdered product.

Many mushrooms are known to have medicinal properties. For example, themaitake mushroom (Grifola frondosa) contains grifolan, an importantbeta-glucan polysaccharide and has been shown to activate macrophages,an important component of the immune system. Laboratory studies haveshown that maitake extract can block the growth of cancer tumors andboost the immune function of mice with cancer.

It has also been found that shiitake mushrooms possess beneficialproperties. A specific amino acid in shiitake helps speed up theprocessing of cholesterol in the liver. Shiitake also appears to be aformidable cancer fighter. A polysaccharide compound called lentinan hasbeen isolated from shiitake, and in laboratory trials, lentinan appearsto stimulate immune-system cells to clear the body of tumor cells.Shiitake appears to be effective against some of the more seriousviruses, such as HIV and hepatitis B.

Reishi mushrooms have been used in China and Japan for years as amedicine for liver disorders, hypertension, and arthritis, andresearchers have found that reishi has anti-allergic, anti-inflammatory,anti-viral, anti-bacterial, and antioxidant properties. In vitroexperiments also indicate that reishi may help fight cancer tumors.

U.S. Pat. No. 6,747,065 describes methods of producing mushroom myceliarich in trace minerals by culturing the mycelia in a broth to whichsupplements have been added.

It would be desirable to improve or enhance the medicinal properties ofthese and other mushrooms, and shorten the growing time for commercialpurposes.

SUMMARY OF THE INVENTION

The present invention accomplishes the above by providing in a generalaspect a method of increasing the amount of one or more functionalcompounds in mushrooms by growing the mushrooms on a substrate that ishigh in the functional compound or compounds of interest. The term“functional compound”, as used herein, means a compound known to oneskilled in the art to have certain medicinal or other beneficialproperties, such as antioxidant, anti-tumorigenic, antidiabetic,antiviral, and the like. It has been found that growing mushrooms onsubstrates that have high levels of the compounds of interest permitsuptake of the compounds by the mushrooms during the growth cycle, andthese levels are maintained during further processing of the mushrooms.Thus, in one aspect, the present invention provides a method ofincreasing the amount of anthocyanins in medicinal mushroom biomass andmushroom mycelia. The method comprises providing a substrate having oneor more anthocyanins therein and growing mushrooms on the substrate. Inadditional aspects, the present invention provides methods of increasingcertain other compounds in medicinal mushroom biomass and mushroommycelia such as selenium or beta glucans, by providing a substrate highin these compounds and growing the mushrooms on the substrate.

In a further aspect, the present invention provides a method ofdecreasing the growing time of mushrooms, by providing a substrate richin anthocyanins. The mushrooms are grown on the anthocyanin-richsubstrate, and the time to fruiting is decreased as compared to growingtime on substrates not having anthocyanins.

In yet a further aspect, the present invention provides a method ofincreasing the potency of naturally-occurring compounds found inmedicinal mushroom biomass. The method comprises providing a substratehigh in anthocyanins, and growing the mushrooms on the substrate.

In an additional aspect, the present invention provides a method ofincreasing the amount of anthocyanins in dried mushroom biomass,comprising providing dried mushroom biomass, and subjecting the driedmushroom biomass to heat treatment.

These and other aspects of the present invention will be come morereadily apparent from the following figure, detailed description andappended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a flow chart illustrating the culturing of mushrooms accordingto a method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Thus, in one aspect the present invention provides a method ofincreasing the amount of one or more functional compounds in medicinalmushroom biomass and mushroom mycelia by growing mushrooms on asubstrate that is high in the functional compound or compounds ofinterest. Examples of “functional compounds” include compounds known tohave antioxidant properties including, but not limited to, anthocyanins;compounds known to have antitumor properties including, but not limitedto, beta glucans; selenium, ganoderic acid; triterpenes; and cordycepin.

As used herein, the term “mushroom biomass” refers to mushroom mycelia,fruiting bodies, spawn, or other life cycle stage of a mushroom, aloneor in combination with each other or in combination with the substrateon which the mushroom is grown, including the functional substratesdescribed herein. “Medicinal mushroom” refers to the varieties ofmushrooms grown for their desired medicinal properties or compounds, asis known in the art.

Thus, in an additional aspect, the present invention provides a methodof increasing the amount of anthocyanins in medicinal mushroom biomassand mushroom mycelia comprising providing a substrate having one or moreanthocyanins therein; and growing mushrooms on the substrate.

Anthocyanins are well known in the art, and are naturally occurringcompounds that are responsible for the red, purple, and blue colors ofmany fruits, vegetables, cereal grains, and flowers. For example, thecolors of berry fruits, such as blueberries, bilberries, strawberries,raspberries, boysenberries, marionberries, cranberries, are due to manydifferent anthocyanins. Over 300 structurally distinct anthocyanins havebeen identified in nature. Because anthocyanins are naturally occurring,they have attracted much interest for use as colorants for foods andbeverages. Recently, the interest in anthocyanin pigments hasintensified because of their possible health benefits as dietaryantioxidants. For example, anthocyanin pigments of bilberries (Vacciniummyrtillus) have long been used for improving visual acuity and treatingcirculatory disorders. There is experimental evidence that certainanthocyanins and flavonoids have anti-inflammatory properties. Inaddition, there are reports that orally administered anthocyanins arebeneficial for treating diabetes and ulcers and may have antiviral andantimicrobial activities. The chemical basis for these desirableproperties of flavonoids is believed to be related to their antioxidantcapacity. Thus, the antioxidant characteristics associated with berriesand other fruits and vegetables have been attributed to theiranthocyanin content.

Proanthocyanins are another class of flavonoid compounds that are foundin fruits and vegetables and, while being colorless, have antioxidantactivities. As used herein, the term “anthocyanins” will refer tonaturally occurring and synthetic anthocyanins and proanthocyanins, asthose terms are understood in the art.

Suitable substrates for growing mushrooms include grains having highlevels of anthocyanins, as noted by the color of the grain. By way ofexample, certain varieties of corn such as purple corn and black cornhave high levels of anthocyanins. Purple barley and purple or black ricevarieties are also known to contain high levels of anthocyanins.

Any variety of mushroom will benefit from the methods of the presentinvention, although due to the intended end use of the mushrooms thevariety must be edible by humans. Suitable varieties include, but arenot limited to, Coriolus versicolor, Ganoderma lucidum, Schizophyllumcommune, Pleurotus ostreatus, Agaricus blazei, Lentinula edodes,Flammulina velutipes, Grifola frondonsus, and Codyceps sinensis.

In an additional aspect, the present invention provides a method ofincreasing the amount of selenium in medicinal mushroom biomass andmushroom mycelia comprising providing a substrate having seleniumtherein; and growing the mushrooms on the substrate. Substrates suitablefor growing mushrooms and having high levels of selenium include, butare not limited to, certain varieties of wheat such as triticumteranicum.

In yet a further aspect, the present invention provides a method ofincreasing the amount of beta-glucan in medicinal mushroom biomass andmushroom mycelia comprising: providing a substrate having beta glucantherein; and growing the mushrooms on the substrate. Beta-glucans are afamily of polysaccharides known in the art for having antitumorproperties, cholesterol lowering ability, and other beneficialproperties. Substrates having high levels of beta-glucans include, butare not limited to, certain varieties of barley such as waxy hullessbarley.

In a further aspect, the present invention provides a method ofdecreasing the growing time of mushrooms, by providing a substrate richin anthocyanins. Suitable substrates are those as described above. Themushrooms are grown on the anthocyanin-rich substrate, and the time tospawning and fruiting is decreased as compared to growing time onsubstrates not having anthocyanins. It has been found that the growingtime can be decreased by 20-25%, using the methods of the presentinvention. This provides a significant advantage and benefit in thecommercial production of mushrooms.

In an additional aspect, the present invention provides a method ofincreasing the potency of naturally-occurring medicinal compounds inmushrooms comprising: providing a substrate high in anthocyanins, andgrowing the mushrooms on the substrate. Preferably, the substrate ispurple or black corn.

As used herein, the term “increased potency” means increased levels oramounts of the naturally occurring medicinal compounds found inmushrooms. It has been found that growing mushrooms on substrates highin anthocyanins such as purple corn or other purple or black pigmentedgrain has the unexpected benefit of increasing the amounts of certaindesirable compounds already present in medicinal mushrooms, including,but not limited to, triterpenes, ganoderic acid, cordycepin, adenosine,and hydroxyethyl-adenosine.

The substrate is prepared for each individual spawn bag. The spawn bagsare designed with a high efficiency particulate air (HEPA) filter andcan be autoclave heat sterilized. The HEPA filter on the spawn bagallows the bag to breathe and protects the substrate from contamination.Each spawn bag contains 5 pounds of prepared substrate consisting of abatch mix of about 3 pounds grain with about 2 pounds water. Theunsealed spawn bags are then autoclave steam sterilized up to atemperature of about 250° F. (approximately 15 psig) for a period ofabout 4-5 hours. The cook time is the time that steam is supplied andshutdown to the autoclave. HEPA-filtered clean air is then applied forrapid cooldown of the autoclave. The cooking time is determined bymonitoring the inner core temperatures of the spawn substrate. Followingautoclaving to sterilize the substrate, the spawn bags are theninoculated with spawn. The spawn bags are then heat sealed and the bagsgently tumbled by machine or by hand to evenly distribute the spawnthroughout the substrate. Thorough mixing may take a few seconds to aminute. Clean room conditions must be maintained during the process toprevent contamination of the substrate. The spawn bags are allowed tospawn run and mature for a period of about 1-2 months, after which timethe mycelium-enriched spawn bags are harvested. The live (fresh) productcan be sold by individual spawn bags without opening (or harvesting) thebags. If a dry product is desired, the spawn bags are opened and thelive product contents are spread out on dryer trays. The product isevenly distributed across the dryer tray with less than about 1″ height.The dryer trays are loaded into a convection air dryer. The airtemperature of the dryer may be adjusted from ambient room temperatureto about 190° F. Usually the air temperatures are set to control around150-170° F. and drying times are automatically set from 16-20 hours. Theair temperatures can be set lower, requiring longer dry times. Freezedrying or vacuum drying may be used as alternative drying methods. Thefinal dried product is tested to have a less than or equal to about 5%by weight moisture content. Each spawn bag initially weighing 5 pounds(water and grain mix) is designed to produce slightly greater than 1 kgof dry product.

The dry product is powdered using a grinder with 40 mesh or finerscreen. The powder is then packaged into 3 kg plastic bags and the bagsare sealed. The bags are then autoclaved for final sterilization for upto approximately 15 hours, at temperatures between about 200° F. toabout 270° F. (approximately 15 psig), depending on the amount ofmoisture removed during the drying step and other factors. Due to thelow moisture content of the dry product, sterilization will take longer.Since the bags are sealed prior to sterilization, regulated air pressureis added to the autoclave during steam shutdown to avoiddepressurization of the autoclave. This will prevent bags from beingdamaged due to the core temperatures in the bag unable to cool as fastas the pressure can decline based on temperature and pressurethermodynamic principles. Once the product is cooled, the product can beremoved from the autoclave.

Surprisingly, it has been found that the final heat treatment stepincreases the amount of anthocyanins present in the dried mushroombiomass. Without wishing to be bound by any theory, it is thought thatthe heat treatment improves the bioavailability of the anthocyanins byreleasing them from a bound form. Thus, in an additional aspect, thepresent invention provides a method of increasing the amount ofanthocyanins in dried mushroom biomass, comprising providing driedmushroom biomass, and subjecting the dried mushroom biomass to heattreatment. Preferably, the heat treatment is carried out for a period ofup to about 15 hours, although shorter time intervals may increase theamount of anthocyanins to a lesser extent. Also preferably, the heattreatment is carried out at temperatures of between about 200° F. toabout 270° F., depending on the moisture content of the dried mushroombiomass and other factors, as will be understood by one skilled in theart. Autoclaving or any other suitable method of heat treatment can beused.

EXAMPLES

The following examples are intended to illustrate the invention andshould not be construed as limiting the invention in any way.

Specimens of Ganoderma lucidum were grown on purple corn by the methodsknown in the art and generally described above and shown in the flowchart in FIG. 1. Sample A, Ganoderma lucidum (GL) grown on purple cornwas tested prior to the final sterilization/heat treatment step for thepresence of anthocyanins. The results are shown in Table 1. TABLE 1Ganoderma lucidum (grown on purple corn) GL-Sample A Analyte Result UnitPelargonidin 0.0022 mg/g Peonidin 0.0018 mg/g Cyanidin 0.0001 mg/gPetunidin 0.0001 mg/g Malvidin 0.0000 mg/g Delphinidin 0.0001 mg/g

Sample B was prepared as above, including the final sterilization/heattreatment for about 10 hours at 250° F. Results are presented in Table2. TABLE 2 Ganoderma lucidum (grown on purple corn) GL-Sample B AnalyteResult Unit Pelargonidin 0.0134 mg/g Peonidin 0.0579 mg/g Cyanidin0.0121 mg/g Petunidin 0.0203 mg/g Malvidin 0.0065 mg/g Delphinidin0.0078 mg/g

Table 3 shows the results of live reishi antler (the fruiting body ofGanoderma lucidum) grown on purple corn, with no further processing. Ascan be seen in these tables, anthocyanins were present in all threesamples grown on purple corn. Mushrooms grown on other nonpigmentedgrains such as rice have undetectable levels of anthocyanins.Additionally, the heat treatment appears to increase the bioavailabilityof the anthocyanins in the mushrooms. TABLE 3 Ganoderma lucidum (Reishiantler grown on purple corn) GL-Sample C Analyte Result UnitPelargonidin 0.0086 mg/g Peonidin 0.0379 mg/g Cyanidin 0.0155 mg/gPetunidin 0.0024 mg/g Malvidin 0.0011 mg/g Delphinidin 0.0193 mg/g

All anthocyanin analysis was performed by methodology employed byKrawczyk, U., Petri, G., “Application of RP [reversed-phase]-HPLC andspectrophotometry in standardization of bilberry anthocyanoside extract”as published in Arch. Pharm. 1992, 325 (3), pp. 147-149 using Partisil10 ODS (25 cm*4.6 mm) with gradient elution (0.5 ml min-1) with formicacid—acetonitrile (100:0 to 0:1 in 60 min then held to 90 min) anddetection at 280 nm. Identification/quantification by external standardsmethod; standard obtained from Sigma-Aldrich.

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details of the presentinvention may be made without departing from the invention as defined inthe appended claims.

1. A method of increasing the amount of selenium in medicinal mushroombiomass and mushroom spawn comprising: providing a substrate havingselenium therein; and growing mushrooms on the substrate.
 2. The methodof claim 1, wherein the substrate is a variety of wheat containingselenium.
 3. The method of claim 2, wherein the wheat is triticumteranicum.
 4. A method of increasing the amount of beta-glucan inmedicinal mushroom biomass and mushroom spawn comprising: providing asubstrate having beta glucan therein; and growing mushrooms on thesubstrate.
 5. The method of claim 4, wherein the substrate is waxyhulless barley.
 6. A method of increasing the amount of anthocyanins indried mushroom biomass comprising: providing dried mushroom biomass; andsubjecting the dried mushroom biomass to heat treatment.
 7. The methodof claim 6, wherein the heat treatment is carried out for a period of upto about 15 hours.
 8. The method of claim 6, wherein the heat treatmentis carried out at a temperature of between about 200° F. to about 270°F.
 9. The method of claim 6, wherein the heat treatment is carried outin an autoclave.